Aromatic prenyltransferase mutant, method for constructing recombinant bacteria for its expression and recombinant bacteria constructed therefrom
A technology of isopentenyl and transferase, applied in the field of bioengineering, can solve the problems of low production stability, harsh conditions, high cost of chemical synthesis, etc., and achieve the effect of strong industrial production and increased yield
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Embodiment 1
[0072] Example 1: The genes required for the MVA pathway were introduced into the genome of Escherichia coli BW25113 by λ-RED homologous recombination to obtain Chassis bacteria with high GPP-producing ability.
[0073] In order to construct a chassis microorganism that efficiently synthesizes cannabinoids such as CBGA, sequence 3 (SEQ ID No.: 1) and sequence 4 (SEQ ID No.: 2) were first synthesized.
[0074] figure 1 Schematic diagram of the genetic elements contained in sequence 3. Such as figure 1 As shown, sequence 3 contains CmR, PMD, MK and idi, wherein CmR is a screening marker, PMD: MVA diphosphate decarboxylase (MVA diphosphate decarboxylase), MK: MVA kinase (MAV kinase), IDI: IPP isomerase, arm1 and arm2 is the BW25113 genome sequence;
[0075] figure 2 Schematic diagram of the genetic elements contained in sequence 4. Such as figure 2 As shown, sequence 4 includes aadA, ERG20, PMK and PMD, aadA is a screening marker with FRT sites at both ends, ERG20: GPP sy...
Embodiment 2
[0112] Example 2: Construction of a plasmid comprising the coding sequence of an aromatic prenyltransferase (NphB) mutant
[0113] First construct the backbone plasmid: p15A-BsaI (SEQ ID NO.: 26) (see Figure 7 ).
[0114] Then, using the golden gate ligation method, the synthetic CBGAS coding genes (sequences of CBGAS-1, CBGAS-2, CBGAS-3 and CBGAS-4 synthesized below) were respectively replaced with the sequence between the BsaI sites in the p15A-BsaI plasmid to obtain p15A - CBGAS-1, pl5A-CBGAS-2, pl5A-CBGAS-3, pl5A-CBGAS-4 plasmids. Wherein, the reaction system is shown in Table 1 below:
[0115] Table 1
[0116]
[0117] The reaction conditions are as follows in Table 2:
[0118] Table 2
[0119]
[0120] After the reaction was completed, 10 μl of the reaction solution was taken to transform Escherichia coli DH5α (purchased from Nanjing GenScript Biotechnology Co., Ltd.) to complete the construction of the plasmid. Constructed plasmids such as Figure 9 shown....
Embodiment 3
[0124] Example 3: Plasmid Transformation and Recombinant Bacteria Fermentation Production of CBGA
[0125] The p15A-CBGAS-1, p15A-CBGAS-2, p15A-CBGAS-3, p15A-CBGAS-4 plasmids constructed in Example 2 were respectively transformed into the chassis bacteria obtained in Example 1 (sequence 1 and sequence 2 BW25113) to obtain recombinant bacteria for CBGA synthesis, and the recombinant bacteria were named: CZ-1, CZ-2, CZ-3 and CZ-4. Recombinant bacteria were processed separately according to the following method, and three parallel samples were made for each recombinant bacteria.
[0126] Fermentation and sample preparation:
[0127] 1. Inoculate the recombinant bacteria into 3mL LB liquid medium, culture overnight at 37°C, 220 rpm, about 14 hours, and the final OD600 value reaches 2-3;
[0128] 2. Add ZYM medium to 24 deep-well plates, add 2ml to each well;
[0129] 3. Transfer the bacterial solution in step 1 to the ZYM medium in step 2, and the OD600 after transfer is 0.01; ...
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